ABSTRACT
High Resolution Transmission Electron Microscopy (HRTEM) studies performed in R. M. Brown laboratory In the Department of Botany at the University of Texas at Austin have focused primarily on the three projects covering the imaging of: (a) single walled carbon nanotubes; (b) T-7 bacteriophage DNA and associate proteins; and (c) cellulose synthase molecules from Acetobacter xylinum. The goal of these studies was to contribute to a better understanding of the high resolution transmission electron microscope in our lab and to observe for the first time the structures of several molecules important in chemistry and biology.
INTRODUCTION
Transmission electron microscopy was developed to circumvent the resolution limits of light microscopes imposed by the wavelength of light. Originally, electron microscopes were used for finding isolated defects in bulk materials, but as resolution became better, these microscopes were used to study biological molecules. High resolution transmission electron microscopy (HRTEM) studies then appeared in all fields of biology and chemistry becoming an integral part of scientific research in these areas.
Our lababoraty focuses mainly on cellulose, and a Philips 420 transmission electron microscope (TEM) is used in conjunction with other laboratory techniques to study cellulose production in vitro and in vivo. This summer, studies were conducted to test the limits of the TEM and its usefulness in diverse areas relating to chemical structure and macromolecular structure in biology. Observations were made of T7 bacteriophage, single-walled carbon nanotubes, and cellulose synthase from Acetobacter xylinum.
MATERIALS AND METHODS
In our laboratory, a Philips 420 transmission electron microscope is used, and for observation in this microscope, specimens are placed on copper mesh grids coated with a thin formvar film which has a minimal electron density and is relatively stable under the low dose or the electron beam (1-2 microamps). Usually, the sample is sonicated then a drop is placed on the grid for 1 minute. If needed, the specimen is then negatively stained with uranyl acetate (UA). Observations are made with a working voltage of 100kV or 120kV at TEM instrument magnifications ranging from 3000X to 750000X. Electron images are converted to high resolution photon images using a YAG crystal detector of the Gatan imaging system. Magnifications on the Gatan monitor is 21.6X the instrument magnification. The live image is then sent to a Kontron IBAS imaging system where it is digitized at a final screen magnification approximately 43.2X over the instrument magnification. The TV magnification is not a limiting factor. Images are processed in the IBAS system to enhance contrast and remove background noise. Time/course images allowed an assessment of exposure to the electron beam. The TEM was routinely calibrated to better than 3.35Å resolution by focusing and stigmating on a graphite lattice. From these calibrations, measurements of observed specimens could be taken.
CONCLUSIONS
In 1998, the potential of the Philips 420 TEM in our lab will continue to be intensively explored. Using HRTEM with final magnifications exceeding 19 million times, electron diffraction, and sophisticated image processing techniques, it has been demonstrated that the TEM is a magnificent tool for studying areas of chemistry, microbiology, botany, and molecular biology. We are planning a detailed publicaction covering this new approach to HRTEM. We are also imaging such molecules as amorphous cellulose, the acetylcholine receptor, cellobiose dehydrogenase, linear acetylenic carbon, cellulose synthase, and protein transcription factors. We are also interested in the imaging of telomerase and its interaction with DNA, as well as other DNA/protein interactions, including RNA structure, and crown ether structure.
